MDS analysis

Goal: Do an MDS plot using Irene’s SNPs to look for relatedness among plants

Snp calling with freebayes

First, need to call SNPs using all BAM files at once:

working in directory 2019/IreneSnps on whitney

first, sort bams

for f in SNPanalysis/*LT*rmdup.bam
    do 
       newname=`basename $f .bam`_sort.bam
       samtools sort -o $newname --reference SNPanalysis/Pinsaporeference1 $f 
    done

next, assign read groups to bams

input=""

for f in `ls *sort.bam`
   do
      rg=`basename $f _rmdup_sort.bam`
      input="$input -b $f -r $rg -s $rg"
  done

echo $input

bamaddrg $input > LT_rmdup_sort_combined.bam

samtools index LT_rmdup_sort_combined.bam

freebayes -f SNPanalysis/Pinsaporeference1 --no-indels --no-mnps --no-complex LT_rmdup_sort_combined.bam > LT.vcf &

try parallel

ulimit -n 4000
/usr/local/stow/freebayes/scripts/fasta_generate_regions.py Pinsaporeference1.fai 100000 > regions
./freebayes-parallel regions 8 -f Pinsaporeference1 --no-indels --no-mnps --no-complex LT_rmdup_sort_combined.bam > LT.vcf

(note: I edited the freebayes-parallel script so that it would work…)

Freeybayes parallel takes about 12 hours

scp whitney.plb.ucdavis.edu:2019/IreneSnps/LT.vcf.gz ../input/

now analyze vcf in R

library(tidyverse)

[30m── [1mAttaching packages[22m ────────────────────────────────────────────────────────────────── tidyverse 1.2.1 ──[39m
[30m[32m✔[30m [34mggplot2[30m 3.0.0     [32m✔[30m [34mpurrr  [30m 0.2.5
[32m✔[30m [34mtibble [30m 1.4.2     [32m✔[30m [34mdplyr  [30m 0.7.6
[32m✔[30m [34mtidyr  [30m 0.8.1     [32m✔[30m [34mstringr[30m 1.3.1
[32m✔[30m [34mreadr  [30m 1.1.1     [32m✔[30m [34mforcats[30m 0.3.0[39m
[30m── [1mConflicts[22m ───────────────────────────────────────────────────────────────────── tidyverse_conflicts() ──
[31m✖[30m [34mdplyr[30m::[32mfilter()[30m masks [34mstats[30m::filter()
[31m✖[30m [34mdplyr[30m::[32mlag()[30m    masks [34mstats[30m::lag()[39m

library(ggrepel)

get the vcf header

vcf.header <- system("zgrep '#C' ../input/LT.vcf.gz",intern = TRUE) 
vcf.header

[1] "#CHROM\tPOS\tID\tREF\tALT\tQUAL\tFILTER\tINFO\tFORMAT\t38LTR\t42LTR\t42LTRR\t43LTR\t43LTRR\t49LTWR\t49LTWRR\t95LTWR\t95LTWRR\t99LTWR"

vcf.header <- vcf.header %>% 
  str_replace("#","") %>% #get rid of the pound sign
  str_split(pattern = "\t") %>% #split on the tabs
  magrittr::extract2(1)
vcf.header

 [1] "CHROM"   "POS"     "ID"      "REF"     "ALT"     "QUAL"    "FILTER"  "INFO"    "FORMAT"  "38LTR"  
[11] "42LTR"   "42LTRR"  "43LTR"   "43LTRR"  "49LTWR"  "49LTWRR" "95LTWR"  "95LTWRR" "99LTWR"

get the data

snps <- read_tsv("../input/LT.vcf.gz", na = c("","NA","."),comment="#",col_names = vcf.header) %>%
  select(-ID, -FILTER) # these are empty columns

Parsed with column specification:
cols(
  CHROM = col_character(),
  POS = col_integer(),
  ID = col_character(),
  REF = col_character(),
  ALT = col_character(),
  QUAL = col_double(),
  FILTER = col_character(),
  INFO = col_character(),
  FORMAT = col_character(),
  `38LTR` = col_character(),
  `42LTR` = col_character(),
  `42LTRR` = col_character(),
  `43LTR` = col_character(),
  `43LTRR` = col_character(),
  `49LTWR` = col_character(),
  `49LTWRR` = col_character(),
  `95LTWR` = col_character(),
  `95LTWRR` = col_character(),
  `99LTWR` = col_character()
)


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snps

filter to keep snps where there is data from all samples

snps <- snps %>% 
  filter({select(., matches("[0-9]")) %>% complete.cases() })
snps

snps <- snps %>% 
  mutate(TOTAL_DEPTH= {str_extract(INFO, "DP=[0-9]*") %>% 
      str_remove("DP=") %>%
      as.numeric() }
  ) %>%
  filter(QUAL >=100,
         nchar(ALT)==1,
         TOTAL_DEPTH > quantile(TOTAL_DEPTH, 0.05),
         TOTAL_DEPTH < quantile(TOTAL_DEPTH, 0.95))
snps

unpack the information differnet samples:

samples <- colnames(snps) %>% str_subset("^[0-9]")
for (s in samples) {
snps <- snps %>%
  separate(!!s, into=paste(s,c("gt","tot.depth","allele.depth","ref.depth","ref.qual","alt.depth","alt.qual","gt.lik"),sep="_"),
           sep=":", convert = TRUE)
}
snps

For the MDS we only need the genotype info

gts <- snps %>%
  select(ends_with("_gt"))
gts

convert this to numeric

geno.numeric <- gts %>%
  lapply(factor) %>% # convert charcters to "factors", where each category is internally represented as a number
  as.data.frame() %>% # reformat
  data.matrix() %>%#  convert to numeric
  t() 
head(geno.numeric[,1:5],10)

            [,1] [,2] [,3] [,4] [,5]
X38LTR_gt      2    2    2    2    2
X42LTR_gt      2    3    2    2    2
X42LTRR_gt     2    2    2    2    1
X43LTR_gt      2    2    2    2    2
X43LTRR_gt     2    2    2    2    2
X49LTWR_gt     2    2    2    2    2
X49LTWRR_gt    3    3    3    3    1
X95LTWR_gt     2    3    3    2    2
X95LTWRR_gt    2    3    3    2    2
X99LTWR_gt     2    2    2    2    2

dim(geno.numeric)

[1]     10 134421

dim(gts)

[1] 134421     10

genDist <- as.matrix(dist(geno.numeric))
dim(genDist)

[1] 10 10

#perform the multi-dimensional scaling
geno.mds <- as.tibble(cmdscale(genDist))
geno.mds$ID <- rownames(geno.numeric) 
geno.mds <- geno.mds %>%
  mutate(ID={str_replace(ID, "W", "N") %>%
       str_replace("RR", "R2") %>%
      str_remove_all("(X|_gt)") },
    response=ifelse(str_detect(ID,"N"), "no recovery", "recovery"))
geno.mds

geno.mds %>%
  ggplot(aes(x=V1, y = V2, label=ID, color=response)) +
  geom_point() +
  geom_text_repel(show.legend=FALSE, direction="y")

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